ABSTRACT Sarcoidosis is a chronic disease of unknown etiology characterized by the formation of non-necrotizing epithelioid granulomas in various organs, especially in the lungs. The lack of an adequate animal model reflecting the pathogenesis of the human disease is one of the major impediments in studying sarcoidosis. In this report, we describe ApoE-/- mice on a cholate-containing high-fat diet that exhibit granulomatous lung inflammation similar to human sarcoidosis. Histological analysis revealed well-defined and non-necrotizing granulomas in about 40% of mice with the highest number of granulomas after 16 weeks on a cholate-containing high-fat diet. Granulomas contained CD4+ and CD8+ T cells, and the majority of the cells in granulomas showed immunoreactivity for the macrophage marker Mac-3. Cells with morphological features of epithelioid cells expressed angiotensin-converting enzyme, osteopontin, and cathepsin K, all characteristics of epithelioid and giant cells in granulomas of human sarcoidosis. Giant cells and nonspecific inclusions such as Schaumann's bodies and crystalline deposits were also detected in some lungs. Granulomatous inflammation resulted in progressive pulmonary fibrosis. Removal of cholate from the diet prevented the formation of lung granulomas. The observed similarities between the analyzed mouse lung granulomas and granulomas of human sarcoidosis, as well as the chronic disease character leading to fibrosis, suggest that this mouse model might be a useful tool to study sarcoidosis.

nodes, and one was from skin/nose. Table 1 summarizesthe gender, age, and anatomical origin of the samples.Statistical AnalysisStudent’s t-tests were performed, and data are pre-sented as means ? SD. Significance was concludedwhen P ? 0.05.ResultsCholate-Containing High-Fat Diet Inducesthe Formation of Granulomas in Lungs ofApoE?/?Mice with Characteristic Features ofGranulomas in Human SarcoidosisThe histopathological hallmark of sarcoidosis is the pres-ence of non-necrotizing epithelioid cell granulomas withcompact appearance and sharp boundaries from thesurrounding lung tissues.16ApoE?/?mice on CHFD de-veloped well-formed granulomas in lungs that were dif-fusely distributed without angio- or bronchocentricity(Figure 1A). Granulomas were present in lungs of around40% of the mice, and their numbers increased fromaround 12 at 8 weeks on CHFD to more than 30 per lungsection after 16 weeks of diet (Figure 1B).The core of the granulomas was formed by epithelioidcells. These cells were distinguished by their large sizeand polygonal shape with prominent cytoplasm (Figure 1,C and D). The majority of lungs granulomas were visibleas dark ball-like structures because of a high collagencontent as revealed by trichrome staining (Figure 1C),whereas others had a lighter appearance with easilyrecognizable epithelioid cells (Figure 1D). These dif-ferences may reflect different stages of granulomamaturation.The activation of T-cells is mandatory for the develop-ment of granulomas.7In sarcoidosis, CD4?lymphocytesare predominant and a smaller number of CD8?T-cellsare typically present in the periphery of granulomas.12,17We observed a similar distribution of CD4?and CD8?Figure 3. In some mouse lungs, Schaumann’sbodies (A, arrows) and crystalline structures (B)were recognizable by a dark brown appearance intrichrome stained sections. Crystals were often ob-served inside of multinucleated giant cells withstrong cathepsin K staining (C; red: cathepsin K;blue: nuclei and autofluorescence of crystals).Multinucleated giant cells were also present inside(D and E, arrows), and they were also cathepsinK-positive (F; red: cathepsin K; blue: nuclei). ThepresenceofiBALTsinbronchovascularbundles(Gand H, arrows) is shown. Occasionally granulo-mas were observed inside of blood vessels (I,arrow). The percentage of lung area occupied byiBALT (J) is shown. iBALTs consisted predomi-nantly of CD8-cells and were attached to bloodvessels (K; red: CD8; green: smooth muscle cell?-actin). Dense accumulation of CD8?cells iniBALT and fewer Mac-3-positive cells in granulo-mas (L; red: CD8; green: Mac-3) are shown. De-creased amount of CD4?(M; red: CD4) and theprevalence of CD8?cells in iBALT (N; red: CD8)are shown. CD4/CD8 ratio in BAL fluid was de-creased after 16 weeks and increased after 19weeksofCHFDwhencomparedwithcontrolmice(O). Scale bars: 20 ?m (A–F); 130 ?m (G); 65 ?m(H and I); 30 ?m (K–N). *P ? 0.05.Mouse Sarcoidosis ModelAJP March 2010, Vol. 176, No. 31151

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cells (Figure 2, A and B) present in 5% and 3.3% of thearea of granulomas, respectively (Figure 2H). Fibroblastswere observed in the perimeter of some granulomas(Figure 2C). Nonlymphoid mononuclear cells were thedominant cellular constituents in all granulomas. Immu-nostaining for macrophage marker Mac-3 showed thatabout 45% of the granuloma-encompassing areas wereMac-3-positive (Figure 2, D and H) with the strongeststaining at the periphery. On the contrary, proteins suchas ACE, osteopontin, and cathepsin K revealed strongexpression in large cells at the core of the granulomas(Figure 2, E–G). These three proteins are highly ex-pressed in epithelioid and giant cells of human granulo-mas of sarcoidosis.18–21Osteopontin-stained areas oc-cupied 23% of the total granulomas area (Figure 2H). Thepredominant expression of epitheloid cell-specific pro-teins such as ACE, osteoponin, and cathepsin K in gran-ulomas is clearly visible in Figure 2, I–K, whereas theexpression of macrophage marker, Mac-3, is not limitedto granulomas (Figure 2L). Osteopontin and cathepsin Kstaining showed similar distribution in granulomas butonly a weak colocalization with the macrophage marker,Mac-3 (Figure 2, F and G).In addition to granulomas, some mice have shown thepresence of inclusions such as Schaumann’s bodies andcalcium oxalate crystals that are frequently observed inlungs of patients with sarcoidosis.17Schaumann’s bodieshave a conchoidal shape and contain calcium carbonate,iron, and oxidized lipids that are responsible for their darkand multihued appearance (Figure 3A). Schaumann’sbodies have shown similarities in shape to epithelioidcells suggesting they originate from this cell type. Cal-cium oxalate crystals with iron accumulation can occurboth inside and outside of Schaumann’s bodies and giantcells.17,22We observed crystals with brown appearance,most likely because of the presence of calcium oxalateand iron, close to Schaumann’s bodies (Figure 3B). Theywere also found inside and adjacent to giant multinucle-ated cells that were cathepsin K-positive (Figure 3C).However, Schaumann’s bodies and crystal inclusions arenot considered to be specific for sarcoidosis.Similar to human sarcoidosis, some granulomas con-tained multinucleated giant cells (Figure 3, D and E).Occasionally, multinucleated giant cells with strong ca-thepsin K expression were observed outside of granulo-mas (Figure 3F).Granulomas of sarcoidosis tend to localize along bron-chovascular bundles and pulmonary lymphatics, but theyare also present in lung parenchyma.23In our study, wehave not observed any predominant distribution of gran-ulomas, but mice developed iBALTs that were localizedalong bronchovascular bundles and close to blood ves-sels (Figure 3, G, H, and K). They were observed after 8and 16 weeks of CHFD and disappeared after 19 weeks(Figure 3J). Occasionally, the presence of iBALT wasaccompanied by granulomas intruding blood vessels(Figure 3I) that might be similar to granulomatous vascu-litis frequently observed in patients with sarcoidosis.23iBALTs showed high cell densities with a predominanceof CD8?cells that clearly distinguished them from gran-ulomas consisted of predominantly Mac-3-positive cells(Figure 3L). In contrast to CD8, CD4 staining was ob-served only in a few cells in iBALTs (Figure 3, M and N).Flow cytometry analysis of BAL fluids revealed a two-fold reduction of the CD4/CD8 ratio at 16 weeks of dietwhen compared with their littermates on the normal diet(Figure 3O). After 19 weeks of the cholate-containingdiet, the CD4/CD8 ratio increased about 3.5-fold whencompared with the controls.Extrapulmonary InvolvementSarcoidosis is a multisystem disorder that can affectmany organs. In addition to lungs, we analyzed skin, liver,heart, kidney, stomach, and thymus. At the later stages ofthe experiment (16 weeks of CHFD), mice developedxanthomas predominantly located at the neck and upperback areas where cutaneous lesions have been ob-served in human sarcoidosis.19The thymus in all mice onCHFD revealed a striking hypertrophy (Figure 4A) with aFigure 4. Organ pathology induced by CHFD. Cholate-containing diet inducedhypertrophy in thymus and fibrosis and induced formation of MGCs in thymus,liver, stomach, and skin. After 16 weeks of CHFD, thymus size was significantlyincreased (A; left: 22-week-old mice on normal diet; right: 16 weeks of CHFD).Trichrome-stained sections of thymus in control mice (B) and highly fibroticacellular thymus after 19 weeks on CHFD (C) are shown. Foreign body-typeMGCs were observed in thymus (D) and stomach (F). Lipid-filled cells with twoto three nuclei were seen in liver (E). Mice on prolonged CHFD developed skinlesions with xanthogranulomas (G). Touton-type MGCs with a central andcirculardistributionofnuclei(H,arrow)andforeignbody-likeMGCs(I,arrow)in skin sections were frequently observed. Scale bars: 130 ?m (B, C, and G); 30?m (E); 20 ?m (D, F, H, and I).1152AJP March 2010, Vol. 176, No. 3Samokhin et al

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more than threefold increase in thymus weight comparedwith mice on the normal diet after 16 and 19 weeks (withaverage wet weights of 34 ? 9 mg in the control groupand 113 ? 42 mg and 112 ? 45 mg after 16 and 19weeks of CHFD, respectively). Histological analysis re-vealed the presence of cholesterol clefts and a progres-sive development of fibrotic changes with an accumula-tion of collagen fibers and a honeycombing appearancewith many multinucleated cells present (Figure 4, B–D). Inthe liver, lipid-filled hepatocytes were observed withsome containing two to three nuclei per cell (Figure 4E).As for the thymus, cholesterol clefts, foreign body-typemultinucleated cells, and an accumulation of collagenwere also found in the stomach (Figure 4F). Xanthogran-ulomas were occasionally observed in skin lesions (Fig-ure 4G) with Touton-like and foreign body type multinu-cleated giant cells (Figure 4, H and I). The presence offoreign body type and giant cells with Touton-like appear-ance has been previously described for cutaneous le-sions of sarcoidosis.24,25Extrapulmonary phenotypessuch as those observed in the skin and thymus did notallow us to determine whether a mice has granulomasbecause we did not observe differences in thymus weightand the occurrence of xanthomas between mice with orwithout lung granulomas that were on CHFD. This mightbe related to the resolution and reoccurrence of lunggranulomas as it is described in sarcoidosis.1Progressive Lung FibrosisWith aging, epithelioid cell granulomas undergo fibrosisand hyalinization. To estimate the rate of fibrosis, we useda method for standardized quantification of pulmonaryfibrosis in murine models.14Feeding CHFD resulted in aprogressive development of lung fibrosis (Figure 5, A–C).The fibrotic grade tripled from 8 to 19 weeks of CHFD(Figure 5J). After 16 weeks of CHFD, lungs of ApoE?/?mice contained individual isolated fibrotic masses withthe development of hyaline membranes in some areas(Figure 5B). Lung areas with the highest degree of fibroticchanges contained confluent fibrotic masses (Figure 5C).As an additional marker for fibrosis, we determined thehydroxyproline content (biochemical collagen marker) inlungs and found a twofold increase after 16 weeks of HFD(Figure 5K). In some areas of lungs, we observed hon-eycombing structures (Figure 5D), which have been de-scribed in human sarcoidosis.17In early fibrotic stages,myofibroblast foci were observed (Figure 5E) indicativefor extracellular matrix production. Intraalveolar protein-aceous deposits were observed in some lungs providingevidence of acute lung injury (Figure 5F). At the laterstages of the CHFD, granulomas were often surroundedby fibrotic tissue without visible changes in granulomacell composition (Figure 5B). Some granulomas, how-ever, revealed fibrotic changes inside their perimeter andFigure 5. Granulomatous inflammation resultedin progressive pulmonary fibrosis. Lung tissuesection of a 22-week-old mice on normal diet(A), granulomas surrounded by single fibroticmasses and hyaline membranes after 16 weeksof CHFD (B, arrows show hyaline membranes),and confluent fibrotic masses after 9 weeks ofCHFD (C) are shown. Fibrosis with honeycomb-ing appearance (D) is shown. Immunostainingfor smooth muscle cell ?-actin revealed the pres-ence of myofibroblasts foci (E) and the deposi-tion of intraalveolar proteinaceous material as aresult of acute lung injuries (F). In some casesgranulomas were characterized by an unusualhigh cell density (G, asterisk shows area mag-nified in H) with the prevalence of CD8-positivecells (I). Quantification of fibrotic changes14(J)is shown. Hydroxyproline content in lungs (K) isshown. Scale bars: 130 ?m (A–D, F, and G); 20?m (E); 30 ?m (H and I). *P ? 0.05.Mouse Sarcoidosis ModelAJP March 2010, Vol. 176, No. 31153

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were characterized by the presence of cells morpho-logically different from epithelioid cells (Figure 5, Gand H) showing an unusually high amount of CD8?cells (Figure 5I).Dietary Cholate and ApoE Deficiency areEssential for the Development of GranulomasTo reveal the cause responsible for granulomas forma-tion, we compared mice after 16 weeks on HFD or CHFDbecause at this time point we observed the highestamount of granulomas. Diet lacking sodium cholate didnot induce granuloma formation in any of the eight micebut induced lung fibrosis (grade 1.4 ? 0.5) that wascomparable with the fibrosis after 8 weeks on CHFD.Mice on HFD did not show thymus hypertrophy and skinxanthomas as seen on the CHFD diet. Moreover, resultswith wild-type DBA/1J mice on 16 weeks on CHFD did notreveal the presence of lung granulomas (data notshown). This may suggest that the presence of sodiumcholate in HFD and apolipoprotein E-deficiency are es-sential for granuloma formation.Human SarcoidosisImmunohistochemical analysis of human sections withlung sarcoidosis revealed that similar to mouse lunggranulomas, cathepsin K was present in epithelioid andmultinucleated giant cells. Some granulomas containedonly epithelioid cells (Figure 6, A and B), whereas inothers, both types of cells were present (Figure 6, C andD). Cathepsin K-positive multinucleated giant cells, witheither a circular arrangement of nuclei (Langhans type,Figure 6E) or a random distribution of nuclei (foreignbody type, Figure 6F), were easily recognizable in areasof granulomatous inflammation. Similar to mouse lungs,giant multinucleated cells were seen adjacent to inclu-sions with dark brown appearance, most likely becauseof the presence of calcium salts and iron deposits (Figure6, G and H).DiscussionAlthough sarcoidosis has been known for more than 100years, the cause of this disease remains a mystery.5Epithelioid granulomas are a hallmark of sarcoidosis.Their similarities to tuberculoid granulomas resulted inthe hypothesis linking mycobacterial infection with sar-coidosis.6,9However, in our experiment mice were keptin a pathogen-free facility and the granulomas appearedto be non-necrotizing thus eliminating the possible in-volvement of a Mycobacteria infection.Granulomas in human sarcoidosis are formed by mac-rophages, epithelioid cells, lymphocytes, and occasion-ally by multinucleated giant cells and fibroblasts.7,12,17,26The lymphocytes present in granulomas are predomi-nantly CD4?, whereas CD8?cells are usually present insmaller numbers.12,26The cell composition of granulo-mas found in lungs of ApoE?/?mice shows a high simi-larity to human granulomas of sarcoidosis.Epithelioid and giant cells are specialized members ofthe monocyte/macrophage lineage that compose thecore of epithelioid granulomas.27Epithelioid cells have aprominent rough endoplasmic reticulum and a complexGolgi suggesting that epithelioid cells change from aphagocytic to a secretory phenotype. One of the proteinssecreted by epithelioid cells in granulomas of sarcoidosisis ACE, and serum levels of this enzyme have beenshown to be elevated in 60% of patients with sarcoid-osis.18,19Angiotensin II generated by ACE promotes lunginjury and conversion of fibroblasts into myofibroblastsfollowed by the accumulation of collagen.28,29Epithelioidcells in lung granulomas of ApoE?/?mice have shownstrong ACE immunoreactivity that, similar to human gran-ulomas, may contribute to the development of fibroticchanges in lungs.Recently, immunohistochemical and in situ hybridiza-tion analyses have shown that cathepsin K might be aFigure 6. Human tissue sections from patients with sarcoidosis show similarcharacteristics regarding cathepsin K expression in granulomas as describedin mice in this report. Epithelioid cells in skin granulomas (A, H&E staining)are cathepsin K immunoreactive (B), lymph node granuloma with epithelioidand giant cells (C, H&E staining) containing strong cathepsin K staining (D).Cathepsin K-positive Langhans-type MGC in lymph node (E) and foreignbody-like MGC in lung (F) are shown. Crystals and inclusions with darkbrown appearance (G, H&E staining) were often seen adjacent or withincathepsin K-positive MGCs (H) in sections with lung sarcoidosis. Scale bars:130 ?m (A, B, G, and H); 30 ?m (C and D); 20 ?m (E and F).1154AJP March 2010, Vol. 176, No. 3Samokhin et al

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marker of macrophage differentiation toward epithelioidand giant cells.20Our results support this notion becausecathepsin K immunoreactivity in epithelioid and giantcells was highly elevated in lung granulomas.In addition to ACE and cathepsin K expression, epi-thelioid cells in murine lung granulomas showed a strongstaining for osteopontin, an extracellular matrix proteinthat is also known as an early T-lymphocyte activationgene-1 and a cytokine that is important for Th1 immun-ity.30,31Th1 immune response mediated by CD4?T-cellsis activated in sarcoidosis and responsible for the initia-tion of granulomas formation.7,8,19Osteopontin is one ofthe most prominently expressed proteins in giant andepithelioid cells in sarcoidosis.21Recently it was shownthat osteopontin can be cleaved by thrombin and sev-eral metalloproteinases.30Thrombin-mediated cleav-age of osteopontin generates amino- and carboxyl-terminalfragments of similar size but with different biological ac-tivities. The amino-terminal fragment appears to enhancecell adhesion and migration, whereas the carboxyl-termi-nal fragment suppresses the ability of full-length os-teopontin to mediate cell adhesion and thus may preventthe development of granulomas in the lungs.32,33On theother hand, full-length osteopontin showed an inhibitoryactivity on foreign body-type multinucleated cell forma-tion.34This may indicate that proteolytic posttranslationalmodifications of osteopontin modulate the formation ofepitheloid and giant cells. The co-expression of os-teopontin and cathepsin K in mouse and human granu-lomas suggests that cathepsin K might be implicated inosteopontin processing and therefore may play a part inthe regulation of sarcoidosis development through thegeneration of physiologically active fragments of os-teopontin and thus promoting epitheloid and giant cellformation.An increase of the CD4/CD8 ratio in BAL fluid is aclinically important marker of sarcoidosis,8but we ob-served a decrease in this ratio after 16 weeks whencompared with control mice. This apparent contradictionto the human situation could be explained by the in-creased presence of iBALT composed predominantly ofCD8?cells at this time point. When iBALT disappearedafter 19 weeks of HFD, the CD4/CD8 ratio increasedalmost fourfold compared with control mice. These re-sults suggest that flow cytometric analysis was unable todetect an increase in the CD4/CD8 ratio because of theprevalence of CD8?cells in iBALT at early stages of ourexperiment, despite the predominance of CD4?cells ingranulomas.The formation of iBALT is rare in mice but occasionallymay be induced in response to pulmonary infections andinflammatory reactions.35iBALT is found in some patientswith interstitial lung disease.36It has been shown thatiBALT can functionally replace the function of conven-tional lymphoid organs and its size and cell compositionvaries widely.37,38Thus, the appearance of iBALT in ourexperiment might be related to the observed hypertrophyand fibrotic changes in thymus.Pulmonary fibrosis occurs in 20% to 25% of patientswith sarcoidosis and it is the most frequent cause ofrespiratory failure that results in deaths related to sarcoid-osis.1,4,19,39Primary effector cells in fibrosis are myofi-broblasts. These cells possess characteristics of bothfibroblasts and smooth muscle cells. Myofibroblasts areextracellular matrix protein-producing cells with a con-tractile phenotype expressing ?-smooth muscle cellactin.40Granulomas in sarcoidosis tend to undergo perigranu-lomatous fibrotic changes, which is in accordance withour observed distribution of myofibroblasts in some gran-ulomas and the development of fibrosis around late stagegranulomas in mice fed on CHFD.The pathogenesis of pulmonary fibrosis in patients withsarcoidosis remains poorly characterized, but it is knownthat the resolution of granulomas or the persistence ofgranulomatous inflammation leading to fibrosis develop-ment depend on the balance between Th1/Th2 cyto-kines.12,19,26In an experiment with cholate-containedhigh-fat diet similar to our experiment, it was shown thatApoE?/?mice had shifted from a Th1 to a Th2 re-sponse.41Th2 cells produce mainly interleukins 4, 10,and 13, which might be responsible for the differentiationof macrophages into epithelioid and giant cells. It wasshown that mouse macrophages in the presence of in-terleukin-4 differentiate into epithelioid cells.42It was fur-ther demonstrated that interleukin-4 induces the alterna-tive activation of macrophages and their fusion intomultinucleated giant cells and subsequently drives thedevelopment of lung fibrosis.43,44It is tempting to spec-ulate that the observed formation of multinucleated giantcells in several organs of mice and granulomatous inflam-mation in lungs during CHFD might be a result of such ashift to a Th2 response.In conclusion, morphological and cell compositionsimilarities between lung granulomas in ApoE?/?mice ona cholate-contained high-fat diet and granulomas ob-served in human sarcoidosis as well as their similar pro-gression into a fibrotic stage suggest that this mousemodel is useful for studying the mechanism of granulo-mas formation. The chronic and progressive granulato-mous inflammation described in this model constitutes animprovement to current models based on acute phase ofgranuloma induction.References1. Nunes H, Bouvry D, Soler P, Valeyre D: Sarcoidosis. Orphanet J RareDis 2007, 2:462. Verleden GM, du Bois RM, Bouros D, Drent M, Millar A, Muller-Quernheim J, Semenzato G, Johnson S, Sourvino G, Olivier D, PietinalhoA, Xaubet A: Genetic predisposition and pathogenetic mechanisms ofinterstitial lung diseases of unknown origin. Eur Respir J Suppl 2001,32:17S–29S3. Lynch JP, 3rd, Ma YL, Koss MN, White ES: Pulmonary sarcoidosis.Semin Respir Crit Care Med 2007, 28:53–744. Nagai S, Handa T, Ito Y, Ohta K, Tamaya M, Izumi T: Outcome ofsarcoidosis. Clin Chest Med 2008, 29:565–5745. Sharma OP: Sarcoidosis: a historical perspective. Clin Dermatol2007, 25:232–2416. Moller DR: Potential etiologic agents in sarcoidosis. Proc Am ThoracSoc 2007, 4:465–4687. Zissel G, Prasse A, Muller-Quernheim J: Sarcoidosis–immunopathogeneticconcepts. Semin Respir Crit Care Med 2007, 28:3–14Mouse Sarcoidosis ModelAJP March 2010, Vol. 176, No. 31155

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[Show abstract][Hide abstract]ABSTRACT: Remodeling of lung tissues during the process of granuloma formation requires significant restructuring of the extra-cellular matrix and cathepsins K, L and S are among the strongest extra-cellular matrix degrading enzymes. Cathepsin K is highly expressed in various pathological granulomatous infiltrates and all three enzymes in their active form are detected in bronchoalveolar lavage fluids from patients with sarcoidosis. Granulomatous inflammation is driven by T-cell response and cathepsins S and L are actively involved in the regulation of antigen presentation and T-cell selection. Here, we show that the disruption of the activities of cathepsins K, L, or S affects the development of lung granulomas in a mouse model of sarcoidosis.
Apolipoprotein E-deficient mice lacking cathepsin K or L were fed Paigen diet for 16 weeks and lungs were analyzed and compared with their cathepsin-expressing littermates. The role of cathepsin S in the development of granulomas was evaluated using mice treated for 8 weeks with a potent and selective cathepsin S inhibitor.
When compared to wild-type litters, more cathepsin K-deficient mice had lung granulomas, but individually affected mice developed smaller granulomas that were present in lower numbers. The absence of cathepsin K increased the number of multinucleated giant cells and the collagen content in granulomas. Cathepsin L deficiency resulted in decreased size and number of lung granulomas. Apoe-/- mice treated with a selective cathepsin S inhibitor did not develop lung granulomas and only individual epithelioid cells were observed.
Cathepsin K deficiency affected mostly the occurrence and composition of lung granulomas, whereas cathepsin L deficiency significantly reduced their number and cathepsin S inhibition prevented the formation of granulomas.

[Show abstract][Hide abstract]ABSTRACT: Sarcoidosis is a granulomatous disease of unknown aetiology. We identified immunological targets for the treatment of pulmonary granulomatosis using a murine model generated with Propionibacterium acnes. Sensitisation and challenge using heat-killed P. acnes and dendritic cells (DCs) were performed to produce pulmonary granulomatosis in C57BL/6 mice. Immunological analyses using ELISA as well as cDNA microarray analysis were used to search for cytokines or chemokines associated with the formation of granulomas in the lungs. Co-administration of P. acnes and DCs reproducibly induced the formation of pulmonary granulomas, which resembled sarcoid granulomas. The cDNA microarray assay demonstrated that the gene expression of CXCL9 and CXCL10, ligands for CXCR3, and of CCL4, a ligand for CCR5, was strongly upregulated during granulomatosis. ELISA confirmed that levels of CXCL9 and CXCL10 as well as T-helper (Th)1 cytokines and chemokines including tumour necrosis factor-α and interferon-γ were elevated in bronchoalveolar lavage fluid (BALF). The blockade of Th1 chemokine receptors using TAK-779, a dual blocker for CXCR3 and CCR5, led to reduced numbers of CXCR3+CD4+ and CCR5+CD4+ T-cells in BALF. Furthermore, administration of TAK-779 ameliorated the granulomatosis. The targeted inhibition of Th1 chemokines might be useful for inhibiting Th1-biased granulomatous diseases, including sarcoidosis.